Systems and methods of discriminating control solution from a physiological sample

ABSTRACT

Described herein are systems and methods for distinguishing between a control solution and a blood sample. In one aspect, the methods include using a test strip in which multiple current transients are measured by a meter electrically connected to an electrochemical test strip. The current transients are used to determine if a sample is a blood sample or a control solution based on at least two characteristics. Further described herein are methods for calculating a discrimination criteria based upon at least two characteristics. Still further described herein are system for distinguishing between blood samples and control solutions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 11/278,333, filed on Mar. 31, 2006, the contents of which areincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Analyte concentration determination in physiological fluids (e.g., atest fluid such as blood or blood derived products such as plasma) is ofever increasing importance to today's society. Such assays find use in avariety of applications and settings, including clinical laboratorytesting, home testing, etc., where the results of such testing play aprominent role in the diagnosis and management of a variety of diseaseconditions. Analytes of interest include glucose for diabetesmanagement, cholesterol for monitoring cardiovascular conditions, andthe like.

A common method for analyte concentration determination assays is basedon electrochemistry. In such methods, an aqueous liquid sample is placedinto a sample reaction chamber in an electrochemical cell made up of atleast two electrodes, i.e., a reference and working electrode, where theelectrodes have an impedance which renders them suitable foramperometric or coulometric measurement. The component to be analyzed isallowed to react directly with an electrode, or directly or indirectlywith a reagent to form an oxidizable (or reducible) substance in anamount corresponding to the concentration of the component to beanalyzed, i.e., analyte. The quantity of the oxidizable (or reducible)substance present is then estimated electrochemically and related to theamount of analyte present in the initial sample.

An automated device, e.g., an electrochemical test meter is typicallyemployed for determining the concentration of the analyte in the sample.Many test meters advantageously allow for an analyte concentration, andusually a plurality of analyte concentrations, to be stored in thememory of the meter. This feature provides the user with the ability toreview analyte concentration levels over a period of time, often timesas an average of previously collected analyte levels, where suchaveraging is performed according to an algorithm associated with themeter. However, to ensure that the system is functioning properly, theuser will occasionally perform test using a control fluid instead ofblood sample. Such control fluids (also referred to as controlsolutions) are generally aqueous solutions having a known concentrationof glucose. The user can perform a test with the control solution andcompare the displayed results with the known concentration to determineif the system is functioning properly. However, once the controlsolution test is performed, the glucose concentration level of thecontrol fluid is stored in the memory of the meter. Thus, when a userseeks to review previous tests and/or the average concentration ofprevious test results, the results may be skewed to the concentration ofthe control fluid analyte level.

Thus, it is desirable to be able to distinguish control solutions andsample fluids during a test. One option is to manually flag the fluidsas either control or test fluids. However automatic flagging would bepreferable since it minimizes user interaction and increasesease-of-use.

As such, there is continued interest in the development of new methodsand devices for use in the determination of analyte concentrations in asample. Of particular interest would be the development of such methodsand devices that include the ability to automatically flag a sample ascontrol fluid or test fluid and to store or exclude measurementsaccordingly. Of particular interest would be the development of suchmethods that are suitable for use with electrochemical based analyteconcentration determination assays.

SUMMARY

The present invention generally provides systems and methods fordistinguishing between a control solution and a blood sample. In oneaspect, described herein, are methods of using a test strip in which apotential is applied and a current is measured. Current values are usedto determine if a sample is a blood sample or a control solution basedon at least one characteristic. Further described herein are methods forcalculating a discrimination criteria based upon at least twocharacteristics. Still further described herein are systems fordistinguishing between blood samples and control solutions.

In one embodiment described herein a method for distinguishing between ablood sample and a control solution sample is disclosed. The methodincludes introducing a sample into an electrochemical cell having firstand second electrodes and applying a first test potential between thefirst electrode and the second electrode. A resulting first currenttransient is then measured. A second test potential is applied betweenthe first electrode and the second electrode and a second currenttransient is then measured. The method can also include applying a thirdtest potential between the first electrode and the second electrode, andmeasuring a third current transient.

Based on the first current transient, a first reference value related tothe quantity of redox species in the sample is calculated. In addition,based on the second and third current transients, a second referencevalue related to reaction kinetics is calculated. The first and secondreference values are then used to determine whether the sample is acontrol sample or a blood sample.

In one aspect, the first reference value is proportional to aconcentration of an interferent in the sample. For example, the firstreference value can be an interferent index calculated based upon atleast one current value from the first current transient. The secondreference values can be a function of a percent completion of a chemicalreaction. For example, the second reference value can be a residualreaction index calculated based upon at least one current value from thesecond current transient and at least one current value from the thirdcurrent transient. In one aspect, the residual reaction index iscalculated based upon a ratio of a second current value and a thirdcurrent value.

In another aspect, the method can perform the step of measuring aconcentration of an analyte in the sample. If the sample is found to bea blood sample, the measured concentration can be stored. Conversely, ifthe sample is found to be a control sample, the measured concentrationcan be flagged, stored separately, and/or discarded.

In one embodiment, statistical classification can be used to determineif the sample is a control solution or a blood sample. For example, anequation representing an empirically derived discrimination line can beused to evaluate the first and second reference values.

In another aspect, an open-circuit potential is applied to theelectrochemical cell before the step of applying the first testpotential. In addition, an open-circuit potential can be applied afterthe step of applying the first test potential.

Further described herein is a system for distinguishing between a bloodsample and a control solution sample, the system including a test stripand a test meter. The test strip comprises electrical contacts formating with the test meter and an electrochemical cell. The test meterincludes a processor adapted to receive current data from the teststrip, and data storage containing discrimination criteria fordistinguishing a blood sample from a control sample based on antioxidantconcentration and reaction kinetics. The discrimination criteria can bederived from an interferent index that is representative of antioxidantconcentration and a residual reaction index that is representative ofreaction kinetics. For example, the discrimination criteria can includean empirically derived discrimination line. The system can furtherinclude a control solution that is substantially devoid of redoxspecies.

Still further described herein is a method for calculating adiscrimination criterion. The discrimination criterion can be programmedinto a test meter for distinguishing between a blood sample and acontrol solution sample. In one embodiment, the method includescalculating an interferent index and a residual reaction index for aplurality of control solution samples and calculating a discriminationcriterion based on a regression of the interferent index and theresidual reaction index for the plurality of control solution samples.

In one aspect, the discrimination criterion is a discrimination line.For example, the method can include plotting an interferent index and aresidual reaction index for a plurality of blood samples and shiftingthe discrimination line towards the plurality of blood samples.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A is a perspective view of an exemplary assembled test strip foruse in method described herein;

FIG. 1B is an exploded perspective view of the test strip of FIG. 1A;

FIG. 1C is an expanded perspective view of a proximal portion of thetest strip of FIG. 1A;

FIG. 2 is a bottom plan view of the test strip of FIG. 1A;

FIG. 3 is a side plan view of the test strip of FIG. 1A;

FIG. 4A is a top plan view of the test strip of FIG. 1A;

FIG. 4B is an expanded partial side view of the proximal portion of thetest strip consistent with arrows 4A-4A of FIG. 4A;

FIG. 5 is a simplified schematic showing a test meter electricallyinterfacing with portions of the test strip;

FIG. 6 shows an example of a potential waveform in which the test meterapplies a series of open-circuit potentials and test potentials forprescribed time intervals;

FIG. 7 shows a current transient generated by the test meter that istesting the test strip with the potential waveform of FIG. 6 with acontrol solution sample (CS, dotted line) and a blood sample (BL, solidline);

FIG. 8 shows the summation of current values at 0.2 and 0.5 seconds fora control solution, plasma, a blood sample with 48% hematocrit, and ablood sample is 77% when a potential of 20 mV was applied;

FIG. 9 is an expanded view of FIG. 7 showing a first test currenttransient and second test current transient for control solution (CS)and blood (BL);

FIG. 10 is a chart showing a non-linear relationship between the % ofsubstrate consumed and the residual reaction index for blood sampleshaving various hematocrit levels and for control solution (diamonds=25%hematocrit blood, squares=42% blood, triangles=60% hematocrit blood,x=control solution; and

FIG. 11 is a chart showing a relationship between an interferent indexand a residual reaction index for a plurality of blood samples(diamonds) and control solution samples (squares).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The subject systems and methods are suitable for use in thedetermination of a wide variety of analytes in a wide variety ofsamples, and are particularly suited for use in the determination ofanalytes in whole blood or derivatives thereof, where an analyte ofparticular interest is glucose. In one embodiment, the subject inventionprovides methods for a test meter to determine whether control solutionor blood has been applied to a test strip. In one aspect, at least twocharacteristics are used to distinguish between a blood sample and acontrol solution. Described herein are structures of an exemplary teststrip embodiment which can be used with the methods and systemsdisclosed herein. Yet further described herein are methods forcalculating a discrimination criterion based upon at least twocharacteristics. Further, described herein are systems fordistinguishing between a blood sample and a control solution.

The subject methods may be used, in principle, with any type ofelectrochemical cell having spaced apart first and second electrodes anda reagent layer. For example, an electrochemical cell can be in the formof a test strip. In one aspect, the test strip includes two opposingelectrodes separated by a thin spacer layer, where these componentsdefine a sample reaction chamber or zone in which is located a reagentlayer. One skilled in the art will appreciate that other types of teststrips, including, for example, test strips with co-planar electrodescould also be used with the methods described herein.

FIGS. 1A to 4B show various views of an exemplary test strip 62 suitablefor use with the methods described herein. Test strip 62 can include anelongate body extending from a proximal end 80 to a distal end 82, andhaving lateral edges 56, 58. The proximal portion of body 59 can includea reaction chamber 61 having electrodes and a reagent, while the distalportion of test strip body 59 can include features adapted forelectrically communicating with a test meter. Physiological fluid orcontrol solution can be delivered to reaction chamber 61 andelectrochemically analyzed.

In the illustrative embodiment, test strip 62 comprises a firstelectrode layer 66 and a second electrode layer 64, with a spacer layer60 positioned therebetween. The first electrode layer 66 can provide afirst electrode 166 and a first connection track 76 for electricallyconnecting the first electrode 166 to a first electrical contact 67.Similarly, second electrode layer 64 can provide a second electrode 164and a second connection track for electrically connecting the secondelectrode 164 with a second electrical contact 63.

In one embodiment, sample reaction chamber 61 is defined by firstelectrode 166, second electrode 164, and spacer 60 as shown in FIGS. 1Ato 4B. Specifically, first electrode 166 and second electrode 164define, respectively, the bottom and top of sample reaction chamber 61.A cutout area 68 of spacer 60 can define the side walls of samplereaction chamber 61. In one aspect, reaction chamber 61 can furtherinclude ports 70 that provide a sample inlet and/or a vent. For example,one of the ports can provide a fluid sample ingress and the other portcan act as a vent.

Reaction chamber 61 can have a small volume. In one embodiment, thevolume ranges from about 0.1 microliters to 5 microliters, preferablyabout 0.2 microliters to about 3 microliters, and more preferably about0.3 microliters to about 1 microliter. To provide the small samplevolume cutout 68 can have an area ranging from about 0.01 cm² to about0.2 cm², preferably about 0.02 cm² to about 0.15 cm², and morepreferably about 0.03 cm² to about 0.08 cm². In addition, first andsecond electrode 166, 164 can be spaced in the range of about 1 micronto 500 microns, preferably between about 10 microns and 400 microns, andmore preferably between about 40 microns and 200 microns. The closespacing of the electrodes can also allow redox cycling to occur, whereoxidized mediator generated at first electrode 166, can diffuse tosecond electrode 164 to become reduced, and subsequently diffuse back tofirst electrode 166 to become oxidized again.

At the distal end of test strip body 59, first electrical contact 67 canbe used to establish an electrical connection to a test meter. Secondelectrical contact 63 can be accessed by the test meter through U-shapednotch 65 as illustrated in FIG. 2. One skilled in the art willappreciate that test strip 62 can include a variety of alternativeelectrical contact configured for electrically connecting to a testmeter. For example, U.S. Pat. No. 6,379,513 discloses an electrochemicalcell connection means, and is hereby incorporated by reference in itsentirety.

In one embodiment, first electrode layer 66 and/or second electrodelayer 64 can be a conductive material formed from materials such asgold, palladium, carbon, silver, platinum, tin oxide, iridium, indium,and combinations thereof (e.g., indium doped tin oxide). In addition,the electrodes can be formed by disposing a conductive material onto aninsulating sheet (not shown) by a sputtering, electroless plating, or ascreen printing process. In one exemplary embodiment, second electrodelayer 64 can be a sputtered gold electrode and first electrode layer 66can be a sputtered palladium electrode. Suitable materials that can beemployed as spacing layer 60 include the variety of insulatingmaterials, such as, for example, plastics (e.g., PET, PETG, polyimide,polycarbonate, polystyrene), silicon, ceramic, glass, adhesives, andcombinations thereof.

Reagent layer 72 can be disposed within reaction chamber 61 using aprocess such as slot coating, dispensing from the end of a tube, inkjetting, and screen printing. Such processes are described, for example,in the following U.S. Pat. Nos. 6,749,887; 6,869,411; 6,676,995; and6,830,934, which are hereby incorporated by reference in their entirety.In one embodiment, reagent layer 72 includes at least a mediator and anenzyme and is deposited onto first electrode 166. Examples of suitablemediators include ferricyanide, ferrocene, ferrocene derivatives, osmiumbipyridyl complexes, and quinone derivatives. Examples of suitableenzymes include glucose oxidase, glucose dehydrogenase (GDH) based onpyrroloquinoline quinone (PQQ) co-factor, GDH based on nicotinamideadenine dinucleotide co-factor, and FAD-based GDH [E.C.1.1.99.10]. Oneexemplary reagent formulation, which would be suitable for makingreagent layer 72, is described in pending U.S. application Ser. No.10/242,951, entitled, Method of Manufacturing a Sterilized andCalibrated Biosensor-Based Medical Device, published as U.S. PublishedPatent Application No. 2004/0120848, which is hereby incorporated byreference in its entirety.

Either first electrode 166 or second electrode 164 can perform thefunction of a working electrode which oxidizes or reduces a limitingamount of mediator depending on the polarity of the applied testpotential of the test meter. For example, if the current limitingspecies is a reduced mediator, then it can be oxidized at firstelectrode 166 as long as a sufficiently positive potential was appliedwith respect to second electrode 164. In such a situation, firstelectrode 166 performs the function of the working electrode and secondelectrode 164 performs the function of a counter/reference electrode. Itshould be noted that unless otherwise stated for test strip 62, allpotentials applied by test meter 100 will hereinafter be stated withrespect to second electrode 164.

Similarly, if a sufficiently negative potential is applied with respectto second electrode 164, then the reduced mediator can be oxidized atsecond electrode 164. In such a situation, second electrode 164 performsthe function of the working electrode and first electrode 166 performsthe function of the counter/reference electrode.

A first step in the subject methods can include introducing a quantityof the fluid sample of interest into test strip 62 which includes firstelectrode 166, second electrode 164 and a reagent layer 72. The fluidsample can be whole blood or a derivative or fraction thereof, orcontrol solution. The fluid sample, e.g., blood, is dosed into samplereaction chamber 61 via port 70. In one aspect, port 70 and/or reactionchamber 61 are adapted such that capillary action causes the fluidsample to fill sample reaction chamber 61.

FIG. 5 provides a simplified schematic showing a test meter 100interfacing with first electrical contact 67 and second electricalcontact 63, which are in electrical communication with first electrode166 and second electrode 164, respectively, of test strip 62. Test meter100 is adapted to electrically connect to first electrode 166 and secondelectrode 164, via first electrical contact 67 and second electricalcontact 63, respectively (as shown in FIGS. 2 and 5). The variety ofknown test meters can be used with the method described herein. However,in one embodiment the test meter includes at least a processor forperforming calculations related to discriminating between blood and acontrol sample and data storage.

As illustrated in FIG. 5, electrical contact 67 can include two prongsdenoted as 67 a and 67 b. In one exemplary embodiment, test meter 100separately connects to prongs 67 a and 67 b, such that when test meter100 interfaces with test strip 62 a circuit is completed. Test meter 100can measure the resistance or electrical continuity between prongs 67 aand 67 b to determine whether test strip 62 is electrically connected totest meter 100. One skilled in the art will appreciate that test meter100 can use a variety of sensors and circuits to determine when teststrip 62 is properly positioned with respect to test meter 100.

In one embodiment, test meter 100 can apply a test potential and/or acurrent between first electrical contact 67 and second electricalcontact 63. Once test meter 100 recognizes that strip 62 has beeninserted, test meter 100 turns on and initiates a fluid detection mode.In one embodiment, the fluid detection mode causes test meter 100 toapply a constant current of 1 microampere between first electrode 166and second electrode 164. Because test strip 62 is initially dry, testmeter 100 measures a maximum voltage, which is limited by the hardwarewithin test meter 100. However, once a user doses a fluid sample ontoinlet 70, this causes sample reaction chamber 61 to become filled. Whenthe fluid sample bridges the gap between first electrode 166 and secondelectrode 164, test meter 100 will measure a decrease in measuredvoltage (e.g., as described in U.S. Pat. No. 6,193,873) which is below apredetermined threshold causing test meter 100 to automatically initiatethe glucose test.

It should be noted that the measured voltage may decrease below apredetermined threshold when only a fraction of sample reaction chamber61 has been filled. A method of automatically recognizing that a fluidwas applied does not necessarily indicate that sample reaction chamber61 has been completely filled, but can only confirm a presence of somefluid in sample reaction chamber 61. Once test meter 100 determines thata fluid has been applied to test strip 62, a short, but finite amount oftime may still be required to allow the fluid to completely fill samplereaction chamber 61.

In one embodiment, once test meter 100 has determined that a fluid hasbeen dosed onto test strip 62, test meter 100 can perform a glucose testby applying a plurality of open-circuit potentials and a plurality oftest potentials to the test strip 62 for prescribed intervals as shownin FIG. 6. A glucose test time interval T_(G) represents an amount oftime to perform the glucose test (but not necessarily all thecalculations associated with the glucose test) where glucose test timeinterval T_(G) can include a first open-circuit time interval T_(OC1), afirst test potential time interval T₁, a second open-circuit timeinterval T_(OC2), a second test potential time interval T₂, and a thirdtest potential time interval T₃. Glucose test time interval T_(G) canrange from about 1 second to about 5 seconds. While two open-circuittime intervals and three test potential time intervals are described,one skilled in the art will appreciate that the glucose test timeinterval can comprise different numbers of open-circuit and testpotential time intervals. For example, the glucose test time intervalcould include a single open-circuit time interval and/or only two testpotential time intervals.

Once the glucose assay has been initiated, test meter 100 switches to afirst open-circuit for a first open-circuit potential time intervalT_(OC1), which in the illustrated embodiment is about 0.2 seconds. Inanother embodiment, first open-circuit time interval T_(OC1) can be inthe range of about 0.05 seconds to about 2 seconds and preferablybetween about 0.1 seconds to about 1.0 seconds, and most preferablybetween about 0.15 seconds to about 0.6 seconds.

One of the reasons for implementing the first open-circuit is to allowsufficient time for the sample reaction chamber 61 to fill or partiallyfill with sample. Typically, at ambient temperature (i.e. 22° C.),sample reaction chamber 61 takes about 0.1 seconds to about 0.5 secondsto completely fill with blood. Conversely, at ambient temperature (i.e.22° C.), sample reaction chamber 61 takes about 0.2 seconds or less tocompletely fill with control solution, where the control solution isformulated to have a viscosity of about 1 to about 3 centipoise.

While control solutions are composed of known components and aregenerally uniform, blood samples can vary in their make-up and/orcomposition. For example, high hematocrit blood samples are more viscousthan low hematocrit blood samples, therefore higher hematocrit bloodsamples require additional time to fill compared with lower hematocritblood samples. Thus, depending on a variety of factors, blood samplefilling time can vary.

After applying the first open-circuit potential, test meter 100 appliesa first test potential E₁ between first electrode 166 and secondelectrode 164 (e.g., −0.3 Volts in FIG. 6), for a first test potentialtime interval T₁ (e.g., 0.15 seconds in FIG. 6). Test meter 100 measuresthe resulting first current transient, which can be referred to asi_(a)(t) as shown in FIG. 7. In one embodiment, first test potentialtime interval T₁ can be in the range of about 0.05 seconds to about 1.0second and preferably between about 0.1 seconds to about 0.5 seconds,and most preferably between about 0.1 seconds to about 0.2 seconds.

As discussed below, a portion or all of the first current transient canbe used in the methods described herein to determine whether controlsolution or blood was applied to test strip 62. The magnitude of thefirst transient current is effected by the presence of easily oxidizablesubstances in the sample. Blood usually contains endogenous andexogenous compounds that are easily oxidized at second electrode 164.Conversely, control solution can be formulated such that it does notcontain oxidizable compounds. However, blood sample composition can varyand the magnitude of the first current transient for high viscosityblood samples will be smaller than low viscosity samples (in some caseseven less than control solution samples) because sample reaction chamber61 may be not be completely filled after 0.2 seconds. An incomplete fillwill cause the effective area of first electrode 166 and secondelectrode 164 to decrease which in turn causes the first currenttransient to decrease. Thus the presence of oxidizable substances in asample, by itself, is not always a sufficient discriminatory factorbecause of variations in blood samples.

After test meter 100 stops applying first test potential E₁, it switchesto a second open-circuit for a second open-circuit time intervalT_(OC2), which in this case is about 0.65 seconds, as shown in FIG. 6.In another embodiment, second open-circuit time interval T_(OC2) can bein the range of about 0.1 seconds to about 2.0 seconds and preferablybetween about 0.3 seconds to about 1.5 seconds, and most preferablybetween about 0.5 seconds to about 1.0 seconds.

One of the reasons for implementing the second open-circuit is toprovide sufficient time for sample reaction chamber 61 to completelyfill, to allow reagent layer 72 to dissolve, and to allow reducedmediator and oxidized mediator to re-equilibrate at the respective firstelectrode 166 and second electrode 164 from the perturbation caused byfirst test potential E₁. Although sample reaction chamber 61 fillsrapidly, second open-circuit time interval T_(OC2) can be sufficientlylong to account for conditions which can cause fill times to increasesuch as low ambient temperature (e.g., about 5° C.) and high hematocrit(e.g., >60% hematocrit).

During first test potential E₁, reduced mediator was depleted at secondelectrode 164 and generated at first electrode 166 to form aconcentration gradient. Second open-circuit potential provides time forthe reduced mediator concentration profile to become closer to the stateimmediately before first test potential E₁ was applied. As will bedescribed below, a sufficiently long second open-circuit potential isuseful because it can allow for glucose concentration to be calculatedin the presence of interferents.

An alternative embodiment test potential E₁′ can be applied between theelectrodes for a duration between when the meter detects that the stripis filling with sample and before a second test potential E₂ is applied.In one aspect, test potential E₁′ is small. For example, the potentialcan be between about 1 to 100 mV, preferably between about 5 mV and 50mV and most preferably between about 10 mV and 30 mV. The smallerpotential perturbs the reduced mediator concentration gradient to alesser extent compared to applying a larger potential difference, but isstill sufficient to obtain a measure of the oxidizable substances in thesample. The potential E₁′ can be applied for a portion of the timebetween detection of fill and when E₂ is applied or can be applied forthe whole of that time period. If E₁′ is to be used for a portion of thetime then an open-circuit could be applied for the remaining portion ofthe time. The combination of number of open-circuit and small voltagepotential applications, their order and times applied is not critical inthis embodiment, as long as the total period for which the smallpotential E₁′ is applied is sufficient to obtain a current measurementindicative of the presence and/or quantity of oxidizable substancespresent in the sample. In a preferred embodiment the small potential E₁′is applied for the entire period between when fill is detected and whenE₂ is applied.

Once second open-circuit time interval T_(OC2) or an equivalent time inthe small potential E₁′ embodiment has elapsed, test meter 100 applies asecond test potential E₂ between first electrode 166 and secondelectrode 164 for a second test potential time interval T₂. Duringsecond test potential time interval T₂, test meter 100 can measure asecond current transient which may be referred to as i_(b)(t). Aftersecond potential time interval T₂ has elapsed, test meter 100 can applya third test potential E₃ between first electrode 166 and secondelectrode 164 for a third test potential time interval T₃, which may bereferred to as i_(c)(t). Second test potential time interval T₂ andthird test potential time interval T₃ can each range from about 0.1seconds to 4 seconds. For the embodiment shown in FIG. 6, second testpotential time interval T₂ was 3 seconds and third test potential timeinterval T₃ was 1 second. As mentioned above, in one aspect, an opencircuit potential time period can be allowed to elapse between thesecond test potential E₂ and the third test potential E₃. Alternatively,the third test potential E₃ can be applied immediately following theapplication of the second test potential E₂. Note that a portion of thefirst, second, or third current transient may be generally referred toas a cell current or a current value.

In one embodiment, first test potential E₁ and second test potential E₂both have a first polarity, and that third test potential E₃ has asecond polarity which is opposite to the first polarity. However, oneskilled in the art will appreciate the polarity of the first, second,and third test potentials can be chosen depending on the manner in whichanalyte concentration is determined and/or depending on the manner inwhich test samples and control solutions are distinguished.

First test potential E₁ and second test potential E₂ can be sufficientlynegative in magnitude with respect to second electrode 164 such thatsecond electrode 164 functions as a working electrode in which alimiting oxidation current is measured. Conversely, third test potentialE₃ can be sufficiently positive in magnitude with respect to secondelectrode 164 such that first electrode 166 functions as a workingelectrode in which a limiting oxidation current is measured. A limitingoxidation occurs when all oxidizable species have been locally depletedat the working electrode surface such that the measured oxidationcurrent is proportional to the flux of oxidizable species diffusing fromthe bulk solution towards the working electrode surface. The term bulksolution refers to a portion of the solution sufficiently far away fromthe working electrode where the oxidizable species was not locatedwithin the depletion zone. First test potential E₁, second testpotential E₂, and third test potential E₃ can range from about −0.6Volts to about +0.6 Volts (with respect to second electrode 164) whenusing either a sputtered gold or palladium working electrode and aferricyanide mediator.

FIG. 7 shows a first, second, and third current transients generated bytest meter 100 and test strip 62 using either a control solution sample(dotted line) or a blood sample (solid line). The control solutionsample contained a 525 mg/dL glucose concentration and the blood samplecontained a 530 mg/dL glucose concentration with a 25% hematocrit. FIG.8 shows an expanded view of first and second current transients in FIG.7. FIGS. 7 and 8 show the resulting current transients when applying thepotential waveform shown in FIG. 6. The description below details howthe current transients can be converted into an accurate glucosemeasurement for the test solution or control solution.

Assuming that a test strip has an opposing face or facing arrangement asshown in FIGS. 1A to 4B, and that a potential waveform is applied to thetest strip as shown in FIG. 6, a glucose concentration can be calculatedusing a glucose algorithm as shown in Equation (Eq.) 1.

$\begin{matrix}{\lbrack G\rbrack = {\left( \frac{i_{2}}{i_{3}} \right)^{p} \times \left( {{a \times i_{1}} - Z} \right)}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

In Eq. 1, [G] is the glucose concentration, i₁ is a first current value,i₂ is a second current value, and i₃ is a third current value, and theterms p, Z, and a are empirically derived calibration constants. Aderivation of Eq. 1 can be found in a pending U.S. application Ser. No.11/240,797 which was filed on Sep. 30, 2005 and entitled “METHOD ANDAPPARATUS FOR RAPID ELECTROCHEMICAL ANALYSIS”, which is herebyincorporated by reference. First current value i₁ and second currentvalue i₂ are calculated from the third current transient and i₃ iscalculated from the second current transient. One skilled in the artwill appreciate that names “first,” “second,” and “third” are chosen forconvenience and do not necessarily reflect the order in which thecurrent values are calculated. In addition, all current values (e.g.,i₁, i₂, and i₃) stated in Eq. 1 use the absolute value of the current.

In another embodiment of this invention, the term i₁ can be defined toinclude peak current values from the second and third current transientsto allow for more accurate glucose concentrations in the presence ofinterferents as shown in Eq. 2.

$\begin{matrix}{i_{1} = {i_{2}\left\{ \frac{i_{pc} - {2i_{pb}} + i_{ss}}{i_{pc} + i_{ss}} \right\}}} & {{Eq}.\mspace{14mu} 2}\end{matrix}$

The term i_(pb) represents a peak current value for second testpotential time interval T₂ and the term i_(pc) represents a peak currentvalue for third test potential time interval T₃. The term i_(ss) is thesteady-state current which occurs after the application of third testpotential E₃. Where Eq. 2 is used, second open-circuit potential timeinterval T_(OC2) is preferably sufficiently long so as to allow Eq. 2 tocompensate for the presence of interferents. When second open-circuitpotential time interval T_(OC2) is too short, second peak current valuei_(pb) can become distorted and can reduce the effectiveness of theinterferent correction calculations. The use of peak current values toaccount for interferents in a physiological sample are described in U.S.Pat. No. 8,529,751, which is incorporated by reference in its entirety.

In one embodiment of this invention, Eq.'s 1 and 2 can be used togetherto calculate a glucose concentration for either blood or controlsolution. In another embodiment of this invention, the algorithm ofEq.'s 1 and 2 can be used for blood with a first set of calibrationfactors (i.e. a, p, and Z) and a second set of calibration factors canbe used for the control solution. When using two different sets ofcalibration factors, the methods described herein for discriminatingbetween a test fluid and a control solution can improve theeffectiveness of the analyte concentration calculations.

In addition, if the test meter determines that the sample type iscontrol solution, the test meter can store the resulting glucoseconcentration of the control sample such that a user can review testsample concentration data separately from control solution data. Forexample, the glucose concentrations for control solutions can be storedin a separate database, can be flagged, and/or discarded (i.e., notstored or stored for a short period of time).

Another advantage of being able to recognize control solutions is that atest meter can be programmed to automatically compare the results (e.g.,glucose concentration) of the test of the control solution with theexpected glucose concentration of the control solution. For example, thetest meter can be pre-programmed with the expected glucose level(s) forthe control solution(s). Alternatively, a user could input the expectedglucose concentration for the control solution. When the test meterrecognizes a control solution, the test meter can compare the measuredcontrol solution glucose concentration with the expected glucoseconcentration to determine if the meter is functioning properly. If themeasured glucose concentration is out of the expected range, the testmeter can output a warning message to alert the user.

In one embodiment, the method described herein uses the presence ofredox species to distinguish a control solution from a blood sample. Themethod can include the step of applying a first test potential E₁′ andusing one or more current values measured during the test potential as adiscriminator. In one aspect, two current values from the first testpotential E₁′ are summed and used as the discriminator. FIG. 8 showsdata for a control solution, plasma, a blood sample with 48% hematocrit,and a blood sample is 77% hematocrit. A potential of 20 mV was appliedfor the first 1 second and current values at 0.2 to 0.5 seconds weresummed. As show in FIG. 8, the summed current values were sufficient todistinguish between a control solution (that was substantially devoid ofinterferents) and blood samples.

In another embodiment, two characteristics of control solution are usedto distinguish control solutions from blood—the presence and/orconcentration of redox species in the sample and reaction kinetics. Themethod disclosed herein can include the step of calculating a firstreference value that is representative of the redox concentration in thesample and a second reference value that is representative of the rateof reaction of the sample with the reagent. In one embodiment, the firstreference value is an interferent oxidation current and the secondreference value is a reaction completion percentage.

In regard to redox species in the sample, blood usually contains variousendogenous redox species or “interferents” such as ascorbic acid anduric acid, as well as exogenously derived interferents such as gentisicacid (gentisic acid is a metabolite of aspirin). Endogenous interferentsare chemical species that can be easily oxidized at an electrode and areusually present in blood within a physiological range for healthyindividuals. Exogenously derived interferents are also a chemicalspecies that can be easily oxidized at an electrode, but are not usuallypresent in blood unless they are inputted into the body via consumption,injection, absorption, and the like.

Control solution can be formulated to be either essentially free ofantioxidants or to have a relatively high interferent concentrationcompared to the interferent concentration in a blood sample. For thecase in which control solution is essentially free of antioxidants, themagnitude of the first current transient should be smaller for controlsolution than for a blood sample as shown in FIG. 9. For the case inwhich control solution has a relatively high concentration ofinterferents, the magnitude of the first current transient should belarger for control solution than for a blood sample (data not shown).

An interferent index can be calculated based on the current valueswithin first current transient. In one embodiment, the interferent indexcan include a summation of current values at two points in time duringthe first current transient. In one example, the current values at 0.3and 0.35 seconds can be used. In another embodiment when a smallpotential E₁ is applied for the entire period between when fill isdetected and E₂, the interferent index is preferably obtained by summingtwo values over a longer period, for example 0.2 seconds to 0.5 seconds.

In general, the interferent index will be proportional to theinterferent concentration and should not substantially depend on theglucose concentration. Therefore, in theory, the test meter should beable to distinguish whether the sample is blood or control solutionbased on the interferent index. However, in practice, using only theinterferent index did not always sufficiently discriminate between bloodand control solution. Although blood typically has a much higherinterferent concentration, there are certain conditions in which thefirst current transient for blood may be attenuated such that it iscomparable to control solution. These conditions include high glucoseconcentration, high hematocrit, low temperature, and incomplete fillingof sample reaction chamber 61. Thus, in one embodiment, an additionalfactor was implemented to enable the test meter to sufficientlydiscriminate between blood and control solution.

The additional factor used for helping discriminate between blood andcontrol solution can be a residual reaction index which is a function ofthe percent of remaining substrate which would have reacted if givenenough time. The residual reaction index relates to the reaction rate inthat a high reaction rate can cause the substrate to be depleted by thereaction. However, the residual reaction index will also depend on theinitial magnitude of the substrate concentration.

Reagent layer 72 can include glucose dehydrogenase (GDH) based on thePQQ co-factor and ferricyanide. When blood or control solution is dosedinto sample reaction chamber 61, glucose is oxidized by GDH_((ox)) andin the process converts GDH_((ox)) to GDH_((red)), as shown in Eq.3.Note that GDH_((ox)) refers to the oxidized state of GDH, andGDH_((red)) refers to the reduced state of GDH.D-Glucose+GDH_((ox))→Gluconic acid+GDH_((red))  Eq. 3

Next, GDH_((red)) is regenerated back to its active oxidized state byferricyanide (i.e. oxidized mediator or Fe(CN)₆ ³⁻) as shown in Eq. 4.In the process of regenerating GDH_((ox)), ferrocyanide (i.e. reducedmediator or Fe(CN)₆ ⁴⁻) is generated from the reaction as shown in Eq.4.GDH_((red))+2Fe(CN)₆ ³⁻→GDH_((ox))+2Fe(CN)₆ ⁴⁻  Eq. 4

In general, the rate of glucose consumption based on Eq.'s 3 and 4 isfaster for control solution than blood. Typically, control solution isless viscous than blood causing the reaction rate of Eq. 3 and 4 to befaster for control solution. Further, the reaction rate is faster forcontrol solution because a portion of the glucose present in the bloodsample must diffuse out of the red blood cells to participate in Eq. 3.This extra step of glucose diffusion out of the red blood cells slowsdown the reaction rate to some measurable degree. FIG. 9 shows that thereaction rate for blood is slower than for control solution as evidencedby the fact that the general absolute slope value (e.g., between 1.2 and4 seconds) for the second current transient is less for the bloodsample. Because of the faster reaction rates in control solutioncompared to blood, the residual reaction index for control solution willgenerally be lower than for blood.

The residual reaction index is a number which is related to the percentof glucose which has not been consumed. A relatively low residualreaction index will indicate that the reactions of Eq.'s 3 and 4 areclose to completion. In contrast, a relatively high residual reactionindex will indicate that the reaction is not close to completion. In oneembodiment, the residual reaction index can be an absolute ratio of acurrent value of third current transient divided by a current value ofthe second current transient, as shown in Eq. 5.

$\begin{matrix}{{abs}\left( \frac{i(4.15)}{i(3.8)} \right)} & {{Eq}.\mspace{14mu} 5}\end{matrix}$

For the denominator of Eq. 5, the current value at 3.8 seconds for thesecond current transient is used. The time of 3.8 seconds was chosenempirically, however, one skilled in the art will appreciate that othercurrent values can be used. In one embodiment, a current value towardsthe end of the second current transient is chosen. During the secondcurrent transient time interval T₂, reduced mediator is oxidized atsecond electrode 164. The current values measured during second currenttransient time interval T₂ were ascribed to ferrocyanide generated byreagent layer 72 at first electrode 166 which then diffused to secondelectrode 164 and became oxidized. It is assumed that reagent layer 72remains close to first electrode 166 after it dissolves in blood causingmost of the ferrocyanide generated by reagent layer 72 to also be closeto first electrode 166. A portion of this generated ferrocyanide candiffuse to second electrode 164.

For the numerator of Eq. 5, the current value at 4.15 seconds was used.Other current values from the third current transient can be chosen,however current value towards the beginning of the third currenttransient are preferred. During the third current transient timeinterval T₃, reduced mediator is oxidized at first electrode 166. Thecurrent values measured during second current transient time interval T₂were ascribed to ferrocyanide generated by reagent layer 72 at firstelectrode 166. Therefore, the current values for the third currenttransient will be larger than the second current transient because mostof the ferrocyanide will be close to first electrode 166 because firstelectrode 166 was coated with reagent layer 72. In addition, thirdcurrent transient will also be larger than second current transientbecause it occurs later in the glucose test allowing for moreferrocyanide to be generated. Thus, the absolute ratio as shown in Eq. 5will be larger if the glucose reaction is still far from completion atthe time of the measurement.

FIG. 10 is a chart showing a non-linear relationship between the % ofsubstrate consumed and the residual reaction index for blood sampleshaving various hematocrit levels and for control solution (diamonds=25%hematocrit blood, squares=42% blood, triangles=60% hematocrit blood,x=control solution). This chart shows that the residual reaction indexis relatively high when the % of substrate consumed is low and that theresidual reaction index is relatively low when the % of substrateconsumed is high for a given sample type/hematocrit value. The % ofsubstrate consumed is derived from a ratio

$\frac{C_{o}}{YSI},$where C_(o) is the substrate concentration at the electrode surface andYSI is the substrate concentration using a standard reference technique.The term C_(o) is derived using the following Eq. 6,

$\begin{matrix}{C_{o} = \frac{i_{ss}L}{2{FAD}}} & {{Eq}.\mspace{14mu} 6}\end{matrix}$

where L is the distance between first electrode 166 and second electrode164, F is Faraday's constant, A is the area of first electrode 166, andD is the diffusion coefficient.

FIG. 11 is a chart showing a relationship between an interferent indexand a residual reaction index for a plurality of blood samples andcontrol solution samples. By plotting the interferent index on theX-axis and the residual reaction index on the Y-axis, a segregationbetween blood and control solution can be observed. A discriminationline can be drawn to determine if the sample is either control solutionor blood. In this embodiment, the interferent index is i(0.3)+i(0.35)and the residual reaction index is

${abs}\left( \frac{i(4.15)}{i(3.8)} \right)$

It should be noted that the times (e.g., 4.15, 3.8) at which the currentvalues where selected for the residual reaction index, were foundempirically. A large number of current ratios were evaluated for theirability to discriminate between blood and control solution samples. Theratio shown in Eq. 5 was selected because it was found to producesignificant separation between blood and control solution samples.

A discrimination line was derived to allow the test meter to determinewhether the sample was control solution or blood. For all of the controlsolution samples tested, the interferent index was plotted versus theresidual reaction index. Next, a line was calculated using linearregression for control solution samples. After calculating an equationfor the line, the perpendicular bias between each data point and theline was calculated. The perpendicular bias represents the shortestdistance between the data point and the line as opposed to a verticalbias which is commonly calculated, A standard deviation was determinedfor all of the perpendicular biases (SD_(perp)). Lastly, the line isshifted 3*SD_(perp) units towards the data points for the blood group.The reason for this approach is that the data for the control solutiongroup show very little scatter and therefore the “99% confidence limit”of the control solution group is well-defined.

In the method described herein, the information obtained from thisstatistical analysis of the residual reaction index and the interferentindex can be used by the test meter to distinguish control solutionsfrom blood samples. The test meter can calculate the interferent indexand residual reaction index and use these values in association with thederived discrimination line (or an equation representing thediscrimination line) to distinguish control solutions from bloodsamples.

Example 1

Preparation of control fluid is disclosed below. The prepared controlfluid was used in the experiments which produced the data illustrated inFIGS. 7 and 11.

Citraconic acid Buffer Component 0.0833 g

Dipotassium citraconate Buffer Component 1.931 g

Methyl Paraben Preservative 0.050 g

Germal II Preservative 0.400 g

Dextran T-500 Viscosity Modifier 3.000 g

Pluronic 25R2 Wicking Agent 0.050 g

1-[(6-methoxy-4-sulfo-m-tolyl)azo]-2-naphthol-6-sulfonic acid disodiumsalt Dye (FD&C Blue No. 1) 0.100 g

D-Glucose Analyte 50, 120, or 525 mg

Deionized Water Solvent 100 g

First citraconic buffer pH 6.5±0.1 was prepared by dissolving requiredquantities of citraconic acid and dipotassium citraconate in deionizedwater. Next, Methyl Paraben was added and the solution was stirred untilthe preservative was fully dissolved. Subsequently Dextran T-500, GermalII, Pluronic 25R2 and1-[(6-methoxy-4-sulfo-m-tolyl)azo]-2-naphthol-6-sulfonic acid disodiumsalt were added sequentially, following complete dissolution of thepreviously added chemical. At this point, the pH of the control fluidwas verified, followed by addition of the requisite quantity of glucoseto obtain a low, normal or high glucose level of control fluid. Afterthe glucose was dissolved completely, the control fluid was left at roomtemperature overnight. Finally, the glucose concentration was verifiedusing a Model 2700 Select Biochemistry Analyzer manufactured by YellowSprings Instrument Co., Inc. The dye used in this control solution has ablue color which reduces the possibility of a user confusing controlsolution with blood, which is normally red.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described, except as indicated by the appended claims. Allpublications and references cited herein are expressly incorporatedherein by reference in their entirety.

What is claimed is:
 1. A method for calculating a discriminationcriteria for programming into a test meter for distinguishing between ablood sample and a control solution sample, the method comprising thesteps of: (a) calculating an interferent index and a residual reactionindex for a plurality of control solution samples in which theinterferent index is based upon summing of at least two current valuesmeasured during a first test potential and the residual reaction indexis based upon a ratio of at least two current values measured during asecond test potential and third test potential, respectively; and (b)calculating a discrimination criteria based on a regression of theinterferent index and the residual reaction index for the plurality ofcontrol solution samples.
 2. The method of claim 1, wherein thediscrimination criteria is a discrimination line.
 3. The method of claim2, wherein the method further comprises the step of: plotting aninterferent index and a residual reaction index for a plurality of bloodsamples and shifting the discrimination line towards the plurality ofblood samples.
 4. A method for distinguishing between a blood sample anda control solution sample, the method comprising the steps of: (a)introducing a sample into an electrochemical cell, the electrochemicalcell comprising: (i) two electrodes in a spaced apart relationship; and(ii) a reagent; (b) applying a first test potential, having a firstpolarity, between the electrodes, and measuring cell current; (c)summing at least two current values measured during the first testpotential to generate an interferent index; (d) using the interferentindex to distinguish between a blood sample and a control solutionsample.
 5. The method according to claim 4, wherein the first potentialis in the range of about 1 mV and 100 mV.
 6. The method according toclaim 1, further comprising the steps of: (e) applying a second testpotential, having the first polarity, between the electrodes andmeasuring cell current; (f) applying a third test potential, having asecond polarity opposite of the first polarity between the electrodesand measuring cell current; (g) determining a residual reaction indexbased on at least one current value measured during the second and thirdtest potentials; (h) using the residual reaction index to distinguishbetween a control sample and a blood sample.
 7. The method according toclaim 6, in which residual reaction index is based on a ratio of atleast one current value measured during the second test potential and atleast one current value measured during the third test potential.